Methods for producing nonadherent avian cell lines

A method for producing a nonadherent avian cell line capable of proliferating in a basal medium in the absence of exogenous trophic factors and cytokines, said method comprising culturing embryonic stem cells obtained from blastodermal disks of fertilized eggs in a culture medium, wherein:

• (a) in at least the first passage, said embryonic stem cells are cultured in a primary medium comprising:
• (i) the trophic factor insulin-like growth factor 1 (IGF-1) and the cytokine ciliary neurotrophic factor (CNTF);
• (ii) an inactivated feeder comprising mouse fibroblast STO cells;
• and
• (iii) a basal medium, wherein the basal medium is supplemented with fetal calf serum at an initial concentration of from 8 to 12%;
• (b) the cells obtained in step (a) are further cultured in a primary medium that has been modified by progressively depriving the primary medium of said trophic factors and said cytokines defined in (i) of step (a), wherein the progressive withdrawal of each trophic factor and cytokine is carried out by successive passage of the cells in a culture medium having a lower concentration of at least one of said trophic factors or said cytokines as compared to the culture medium of the prior passage, until the medium is free of all exogenous trophic factors and cytokines;
• and
• (c) the cells obtained in step (b) are inoculated at a high density into a bacteriological dish to produce a nonadherent avian cell line capable of proliferating in a basal medium in the absence of exogenous trophic factors and cytokines.